Strand Displacement Amplification (SDA) utilizes the ability of a restriction enzyme to nick a hemimodified recognition site and the ability of a polymerase to displace a downstream DNA strand to amplify a target nucleic acid (U.S. Pat. No. 5,270,184, hereby incorporated by reference; Walker, et al. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396; Walker, et al. 1992. Nucl. Acids Res. 20, 1691-1696). The target for SDA may be present on fragments of nucleic acid generated by treatment with a restriction endonuclease, or targets appropriate for SDA may be generated by extension and displacement of primers. This second type of target generation and the subsequent steps of the SDA reaction are illustrated in FIG. 1. The target generation process (left side of FIG. 1) produces copies of the target sequence flanked by the nickable restriction sites required for SDA. These modified target sequences are exponentially amplified by repeated nicking, strand displacement, and repriming of displaced strands (right side of FIG. 1). Despite the apparent complexity of FIG. 1, SDA operates under a very simple protocol: double-stranded target DNA is heat denatured in the presence of all reagents except the restriction enzyme and polymerase. Exponential amplification then proceeds at a constant, reduced temperature upon addition of the enzymes, without any further manipulation of the reaction. SDA is capable of 10.sup.8 -fold amplification of target sequences in 2 hours at a constant reaction temperature, usually about 35.degree.-42.degree. C.
Fluoresence Polarization (FP) is a measure of the time-average rotational motion of fluorescent molecules. It has been known since the 1920's and has been used in both research and clinical applications for sensitive determination of molecular volume and microviscosity. The FP technique relies upon changes in the rotational properties of molecules in solution. That is, molecules in solution tend to "tumble" about their various axes. Larger molecules (e.g., those with greater volume or molecular weight) tumble more slowly and along fewer axes than smaller molecules. They therefore move less between excitation and emission, causing the emitted light to exhibit a relatively higher degree of polarization. Conversely, fluorescence emissions from smaller fluorescent molecules, which exhibit more tumbling between excitation and emission, are more multiplanar (less polarized). When a smaller fluorescent molecule takes a larger or more rigid conformation its tumbling decreases and the emitted fluorescence becomes relatively more polarized. This change in the degree of polarization of emitted fluorescence can be measured and used as an indicator of increased size and/or rigidity of the fluorescent molecule.
In fluorescence polarization techniques, the fluorescent molecule is first excited by polarized light. The polarization of the emission is measured by measuring the relative intensities of emission (i) parallel to the plane of polarized excitation light and (ii) perpendicular to the plane of polarized excitation light. A change in the rate of tumbling due to a change in size and/or rigidity is accompanied by a change in the relationship between the plane of excitation light and the plane of emitted fluorescence, i.e., a change in fluorescence polarization. Such changes can occur, for example, when a single stranded oligonucleotide probe becomes double stranded or when a nucleic acid binding protein binds to an oligonucleotide. Fluorescence anisotropy is closely related to FP. This technique also measures changes in the tumbling rates of molecules but is calculated using a different equation. It is to be understood that polarization and anisotropy are interchangeable techniques for use in the present invention. The term fluorescence polarization is generally used herein but should be understood to include fluorescence anisotropy methods. In steady state measurements of polarization and anisotropy, these values are calculated according to the following equations: ##EQU1## where Ipa is the intensity of fluorescence emission parallel to the plane of polarized excitation light and Ipe is the intensity of fluorescence emission perpendicular to the plane of polarized excitation light.
As FP is homogenous, this technique is ideal for studying molecular interactions in solution without interference by physical manipulation. Fluorescence polarization is therefore a convenient method for monitoring conversion of single-stranded fluorescently labelled DNA to double-stranded form by hybridization (Murakami, et al. 1991. Nucl. Acids Res. 19, 4097-4102). The ability of FP to differentiate between single and double-stranded nucleic acid conformations without physical separation of the two forms has made this technology an attractive alternative for monitoring probe hybridization in diagnostic formats. European Patent Publication No. 0 382 433 describes fluorescence polarization detection of amplified target sequences by hybridization of a fluorescent probe to the amplicons or by incorporation of a fluorescent label into the amplification products by target-specific extension of a fluorescently-labeled amplification primer. PCT Patent Publication No. WO 92/18650 describes similar methods for detecting amplified RNA or DNA target sequences by the increase in fluorescence polarization associated with hybridization of a fluorescent probe.
Fluorescence polarization may be monitored as either transient state FP or steady state FP. In transient state FP, the excitation light source is flashed on the sample and polarization of the emitted light is monitored by turning on the photomultiplier tube after the excitation light source is turned off. This reduces interference from light scatter, as fluorescence lasts longer than light scatter, but some fluorescence intensity is lost. In steady state FP, excitation light and emission monitoring are continuous (i.e., the excitation source and photomultiplier tube are on continuously). This results in measurement of an average tumbling time over the monitoring period and includes the effects of scattered light.
The present invention provides FP or fluorescence anisotropy detection methods for use with nucleic acid amplification methods such as SDA. Previously, SDA-amplified target sequences were detected following amplification using .sup.32 P-probes (Walker, et al. 1992 Nucl. Acids Res., supra) or by a sandwich hybridization assay with chemiluminescent signal generation (Spargo, et al. 1993. Molec. Cell. Probes 7, 395-404). Both of these detection formats require separation of free and bound detector probe before the signal can be measured. However, the ability to differentiate free and bound probe using FP without physical separation enables performance of SDA and detection of amplification in a homogeneous, closed system. Furthermore, it has been discovered that SDA and FP detection can be combined in a single step (i.e., real-time amplification and detection), in part as a result of the isothermal nature of SDA. A closed, homogeneous assay reduces operating steps and procedural complexity, as well as providing improved control of the dispersal of amplification products in the laboratory, thereby reducing the potential for false positives due to accidental contamination of samples with target DNA.
Certain of the terms and phrases used herein are defined as follows:
An amplification primer is a primer for amplification of a target sequence by primer extension or ligation of adjacent primers hybridized to the target sequence. For amplification by SDA, the oligonucleotide primers are preferably selected such that the GC content is low, preferably less than 70% of the total nucleotide composition of the probe. Similarly, for SDA the target sequence preferably has a low GC content to minimize secondary structure. The 3' end of an SDA amplification primer (the target binding sequence) hybridizes at the 3' end of the target sequence. The target binding sequence confers target specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease near its 5' end. The recognition site is for a restriction endonuclease which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by Walker, et al. (1992. Proc. Natl. Acad. Sci. and Nucl. Acids Res., supra). The SDA amplification primer generally also comprises additional sequences 5' to the restriction endonuclease recognition site to allow the appropriate restriction endonuclease to bind to its recognition site and to serve as a primer for the polymerase after nicking, as is known in the art. For the majority of the SDA reaction, the amplification primer is responsible for exponential amplification of the target sequence. The SDA amplification primer may also be referred to as the "S" primer, e.g., S.sub.1 and S.sub.2 when a pair of amplification primers is used for amplification of a double stranded sequence. For other amplification methods which do not require attachment of specialized sequences to the ends of the target, the amplification primer generally consists of only the target binding sequence.
A bumper or external primer is a primer used in SDA which anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream primer and its extension product. Bumper primers may also be referred to as "B" primers, e.g., B.sub.1 and B.sub.2 when a pair of bumper primers is used to displace the extension products of a pair of amplification primers. Extension of bumper primers is one method for displacing the extension products of amplification primers, but heating is also suitable.
The terms target or target sequence refer to nucleic acid sequences amplifiable by amplification primers. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified, and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies also serve as amplifiable target sequences by virtue of the fact that they also contain copies of the original target sequences to which the amplification primers hybridize.
Copies of the target sequence which are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the single-stranded copy of a target sequence produced by hybridization of a primer and extension of the primer by polymerase using the target sequence as a template.